For an enlarging group of quantitative analytical methods used in
the clinical chemistry laboratory, external quality-assessment programs
are of crucial importance in guaranteeing the accuracy of the results
produced in the routine laboratory. Presently there is a consensus that
national quality control assessment programs in clinical chemistry
should be based on Reference and Definitive Methods for substrates and
drugs [1,2]. Limitations for the development of Reference Methods exist
mostly because one accepted technique for the design of such methods is
the isotope dilution technique, which requires a final mass
spectrometric analysis at a suitable mass of a fragment of the analyte
and the isotope-modified internal standard [3]. Until now gas
chromatography has been the most widely used separation technique, and
several such methods for the determination of total cholesterol in serum
have been described [4-8]. Attempts have also been made to determine
cholesterol by HPLC [9], and mass spectrometry by direct inlet, after
liquid chromatographic separation and collection of the peak fraction,
has been described [10].

Here we describe our new liquid chromatographyisotope dilution mass
spectrometry (LC IDMS) (1) method for the determination of cholesterol
in serum. The novelty of this method is the separation of the analyte by
HPLC previous to mass spectrometry. The evaporation of the eluent is
done in a particle-beam interface used for coupling the liquid
chromatograph and the mass spectrometer. Finally selective ion
monitoring (SIM) is performed after electron impact (EI) ionization. A
derivatization prior to analysis on the LC-MS instrument is not
required. The results obtained for pooled human sera were compared with
those obtained by a slightly modified variant of the gas chromatography
(GC) IDMS method used by Siekmann [11]. It was not intended to propose a
candidate reference method, but to present an analytical procedure,
which may be worked out to a candidate reference method for the
determination of total cholesterol in serum.

Principles

The isotope-dilution method for the determination of total
cholesterol in serum is based on the addition of identical volume
fractions of the internal standard [25,26,27-[sup.13]
[C.sub.3]]cholesterol to serum samples and calibrators. The serum is
submitted to a basic hydrolysis to convert the cholesterol esters to
free cholesterol. The cholesterol is then transferred to cyclohexane by
liquid-liquid extraction. The cyclohexane phase is taken to dryness. For
the LC IDMS method, the residue containing the cholesterol is dissolved
in ethanol, and this solution is used directly for analysis. The LC
separation is performed on a reversed-phase column. Detection is
performed by spectrophotometric detection at a wavelength of 210 nm and
SIM of the molecular mass ion of cholesterol, m/z = 386, and the
respective molecular mass ion of the internal standard, m/z = 389.

In the case of the GC IDMS method, a derivatization of the
alcoholic groups to trimethylsilyl ethers has to be done prior to
analysis. The GC separation is performed on a nonpolar fused-silica
capillary GC column. In this case, the principal isotope ratio
measurements are made from the ion abundances of the molecular mass ion
of trimethylsilyl cholesterol, m/z = 458, and the respective molecular
mass ion of the internal standard, m/z = 461. Standards are made by
combining pure unlabeled cholesterol and
[25,26,27-[sup.13][C.sub.3]]cholesterol to give one with an unlabeled/
labeled ratio of ~1.0, one standard somewhat lower, and one somewhat
higher. These mixtures are evaporated without previous liquid-liquid
extraction and processed further like the serum samples. To get the best
possible precision, a bracketing technique was applied for both methods
[6].

Materials and Methods

CHEMICALS

The cholesterol used for preparation of the calibrator solutions
was Standard Reference Materials (SRM911b) purity from NIST (obtained
from Promochem, Wesel, Germany). This material has a purity of 99.8%,
(uncertainty 0.1%). The [25,26,27-[sup.13][C.sub.3]]cholesterol used as
internal standard in GC IDMS measurements as well as LC IDMS
measurements was obtained under MS-3501 from IC Chemikalien, Munich,
Germany. The material had a certified 99% isotope enrichment (neither
uncertainty of the isotope enrichment nor the purity of the material was
given by the supplier). N-Methyl-N-(trimethylsilyl)trifluoroacetamide
and silylation-grade pyridine were obtained from Macherey & Nagel.
All other chemicals were of analytical grade and purchased from Merck,
Sigma, and Baker. Deionized water was prepared with a MilliQ apparatus
(Millipore).

SAMPLES

The control materials used for checking accuracy of the methods
were human serum SRM 909 with a certified cholesterol concentration of
1415 [+ or -] 46 mg/L; SRM 909b, grade 1 with a certified cholesterol
concentration of 1464 [+ or -] 18 mg/L; and SRM 909b, grade 2 with a
certified cholesterol concentration of 2353 [+ or -] 30 mg/L from NIST.
The other control materials were Precinorm U from Boehringer Mannheim
with a target value for the concentration of total cholesterol of 1200
mg/L and Kontrollogen-LP from Behringwerke with a target value of 1330
mg/L. Both control materials were based on processed human sera, and the
target value for total cholesterol was determined by use of a GC IDMS
method that was not further specified.

For the method comparison pooled sera were used (n = 28). The pools
were prepared with serum samples taken after analysis in the Central
Laboratory of the Institute of Clinical Chemistry and Pathobiochemistry
at the University Hospital of the Technical University Aachen. Samples
were obtained from the outpatients' departments as well as the
patient care units of the University Hospital. All specimens were
collected in Sarstedt monovettes with separation gel. Serum was obtained
after centrifugation.

INSTRUMENTS AND SETTINGS

LC IDMS. The analysis was performed on a Waters Integrity system
(Waters), consisting of an Alliance 2690 chromatography module, a column
bypass module, a photodiode array detector 996, and a Waters Thermabeam
mass detector, equipped with ion-source working unchangeable in
[El.sup.-] mode. System controlling, data acquisition, and integration
were performed with the Waters Millennium Software, Rel. 2.21.

For chromatography a 150 mm X 2 mm Novapak C18 analytical minibore
column (Waters Chromatography) was used. The eluent consisted of
acetonitrile and isopropyl alcohol (65:35 by vol). The flow rate was 0.3
mL/min. The injection volume was 5 [micro]L for all samples. The
duration of one chromatographic run was 7 min.

The helium flow in the particle-beam liquid chromatography-mass
spectrometry interface was 30 mL/min. The nebulizer was heated to
70[degrees]C, and the temperature of expansion region was set to
90[degrees]C. The pressure in the interface was constantly at 67 Pa. The
temperature of the ion source was set to 220[degrees]C, the El energy
was set to 70 eV, and the pressure in the ion chamber was [less than or
equal to] 0.026 Pa. The voltage settings for the ion optic were 5 V for
the ion volume, -42 V for the extraction lens, -20 V for the prequad,
and -101 V for the exit lens. The multiplier voltage was set to 1980 V.

The measurement was performed in SIM mode at m/z = 386
(cholesterol) and m/z = 389 ([25,26,27[sup.13][C.sub.3]]cholesterol)
with a frequency of 1 scan/s; within one chromatography 420 scans were
performed, and the duration of the elution of the cholesterol peak was
about 50 scans.

GC IDMS. The instrument used was a Fisons MD-800 combined gas
chromatograph-quadrupole mass spectrometer (Fisons Instruments),
equipped with an El source and a GC8000 series gas chromatograph and
autosampler AS800. For instrument controlling and data acquisition the
Fisons MassLab Software Rel. 1.30 was used.

The gas chromatography was performed on a Hewlett-Packard Ultra 1
[0.33 [micro]m, 12 m X 0.32 mm (i.d.)] capillary column (IAS, Leipzig,
Germany). The carrier gas was helium at 690 kPa (100 psi) at a flow rate
of 1 mL/min, the split exit was set at 50 mL/min (1:50), the injector
temperature was 320[degrees]C, the oven temperature was isothermal 280[degrees]C, and the interface temperature was set to 290[degrees]C.
The sample size injected by the AS800 was constantly set to 1.0
[micro]L.

The temperature in the ion source was set to 200[degrees]C, the El
energy was set to 70 eV, and the emission current was set to 235 mA. The
voltage settings for the ion optic were 1.4 V for the ion energy, 0.7 V
for the repeller, 8 V for lens 1, 79 V for lens 2, 7.4 V for low mass
resolution, and 12.2 V for high mass resolution. The multiplier voltage
was set to 500 V.

For SIM mode measurements mass detection was set at m/z = 458 [+ or
-] 0.25 (cholesterol) and m/z = 461 [+ or -] 0.25 ([25,26,27-[sup.13]
[C.sub.3]]cholesterol), the dwell time was set to 0.15 s, and the
channel delay to 20 ms, leading to a measurement frequency of 3 scans/s.
The data acquisition delay time was set to 2.45 min. Within one
chromatography 450 scan were performed, and the duration of the elution
of the peak of the cholesterol derivate was about 30 scans.

PROCEDURES AND MEASUREMENTS

Weighing and pipetting procedures. Cholesterol (SRM 911b) and the
isotopically labeled [25,26,27-[sup.13] [C.sub.3]]cholesterol were
weighed on a microbalance (Mikrowaage 708501, Fa. Sartorius). This
balance has a weight range of 15 mg, and the certified accuracy is 0.5%
at 1 mg and 0.15% at 10 mg, respectively, which was checked with
calibrated weight prior to each use. All other weighing procedures,
including all required calibrations of volumetric devices, were done on
a semimicrobalance (Halbmikrowaage AC 211 S-OCE, Fa. Sartorius). This
balance has a single measuring range up to 210 g with a certified
reproducibility of [less than or equal to] [+ or -]0.1 mg and certified
linearity deviation of [less than or equal to] [+ or -]0.2 mg. All
pipetting procedures were performed with Digital Syringe Series 1700
syringes (Fa. Hamilton). Every volume setting used was calibrated
gravimetrically before use.

Sample preparation for calibrators, control materials, and pooled
sera. The reconstitution of lyophilized control sera was performed as
described previously [1]. The calibrators of cholesterol and
[25,26,27-[sup.13] [C.sub.3]]cholesterol with a concentration of 1 mg/mL
were prepared fresh every day by dissolving 10 mg of solute in 10 mL of
ethanol. To minimize the effects of the varying accuracy of the syringe
with varying pipetting volume, in all cases 100 [micro]L (corresponding
to 100 [micro]g of [25,26,27-[sup.13] [C.sub.3]]cholesterol) of internal
standard were pipetted, so only the volume of the unknown sample or
calibrator had to be varied.

Internal standards. Aliquots of 100 [micro]L of the internal
standard, the labeled [25,26,27-[sup.13][C.sub.3]]cholesterol solution,
were placed in Reacti-vial test tubes. Then aliquots of 75 [micro]L
(standard 1) or 125 [micro]L (standard 2) of unlabeled cholesterol were
added, and the tubes were gently swirled. Two samples were required for
the determination of the isotope ratio in the pure unlabeled and the
labeled cholesterol. For this we placed 200 [micro]L of unlabeled
cholesterol in one vial and 200 [micro]L of the labeled [25,26,27-13
C3]cholesterol in another. The ethanol was removed under a stream of
nitrogen at 60[degrees]C. For LC IDMS measurements the residue was
dissolved in 100 [micro]L of ethanol. For GC IDMS measurements we
dissolved the residue in 50 [micro]L of
N-methyl-N-(trimethylsilyl)trifluoroacetamide/pyridine, and the
derivatization was performed for 30 min at 60[degrees]C.

Control materials, pooled sera. Aliquots of 100 [micro]L of the
internal standard, the labeled [25,26,27-[sup.13][C.sub.3]]cholesterol
solution, were placed in test tubes. Then appropriate aliquots of the
control material or pooled serum were added volumetrically to give an
isotope ratio of ~1.0, and the tubes' contents were gently swirled.

To prepare a set for the total cholesterol determination, we then
added to each of the test tubes 150 [micro]L of an aqueous potassium
hydroxide solution (8.9 mol/L) and 1 mL of ethanol. This mixture was
gently swirled and then heated at 50[degrees]C for 3 h. To check for
complete hydrolysis, the hydrolysis was performed in a separate
experiment by adding 300 [micro]L of the aqueous 8.9 mol/L potassium
hydroxide solution and 1 mL of ethanol, swirling, and finally heating at
50[degrees]C for 6 h. After hydrolysis, 1 mL of deionized water and 2 mL
of cyclohexane were added. After continuous shaking for 5 min the
cyclohexane phase was transferred to Reacti-vials. The samples were
dried and derivatized as described for the internal standard.

Calibration and calculation for the determination of cholesterol in
serum with the GC IDMS and LC IDMS methods. For the measurement of the
unknown samples, each sample of a control material or serum was measured
in triplicate bracketed by triplicate measurements of standard 1
(isotope ratio ~0.75) and standard 2 (isotope ratio ~1.25) in either the
order: lower weight ratio standard, sample, higher weight ratio
standard. This measurement was then repeated in the reversed order. The
three observed intensity ratios were acceptable only if the CV was
<0.5%, then they were averaged. If this could not be achieved, the
measurement of the standard, control, or unknown sample was discarded.
The quantity of analyte in the sample was calculated by linear
interpolation of the measured ratio of the sample between the measured
ratios of the standards with the known weight ratios as described
elsewhere [6]. In every series two values for each control material or
serum sample were obtained by this procedure, and these two values were
averaged.

Results

SPECIFICITY AND DETECTION LIMIT OF THE LC IDMS AND GC IDMS METHODS

The mass spectra of cholesterol and [25,26,27-[sup.13]
[C.sub.3]]cholesterol obtained for LC-MS in scan mode with 1 scan/s,
with the same settings as in the SIM experiments, were identical to
those previously published [10]. The spectra show characteristics
similar to those published earlier [8]. Fig. 1 shows the UV-chromatogram
at a wavelength of 210 nm as well as the SIM chromatograms at the
molecular mass ions m/z = 386 and m/z = 389 for an extract of a serum
sample. The interferences observable in the UV-chromatogram are not
visible in the SIM measurements.

The El ionization was sensitive enough for detection of the
underivatized cholesterol, and the peak of the molecular ion was the
base peak in the mass spectrum; in the case of the
trimethylsilyl-derivatized cholesterol the base peak was at m/z = 329
for the unlabeled cholesterol as expected from previous publications
[12], but the detection limit at m/z = 458 was nearly identical to that
at m/z = 329. The signal-to-noise ratios were comparable, the retention
time of the cholesterol in the LC IDMS method was 5.44 min, and the
retention time of the cholesterol derivative in the GC IDMS method was
4.15 min.

We determined the isotope ratios of the pure unlabeled cholesterol
and the pure [25,26,27-[sup.13][C.sub.3]]cholesterol from the two
samples containing only one type of cholesterol, which were required in
the further calculations. The pure unlabeled cholesterol had a peak
intensity ratio ([A.sub.389]/ [A.sub.386]) of 0.00242, the
trimethylsilyl derivative a peak intensity ratio
([A.sub.461]/[A.sub.458]) of 0.0344; the pure labeled cholesterol had a
peak intensity ratio ([A.sub.386]/[A.sub.389]) of 0.00646, the
trimethylsilyl derivative a peak intensity ratio
([A.sub.458]/[A.sub.461]) of 0.0175.

[FIGURE 1 OMITTED]

INTERFERENCE OF ENDOGENOUS AND EXOGENOUS STEROIDS

We tested the GC IDMS method and the LC IDMS method for the
possible interference of 7-dehydrocholesterol,
5[alpha]-cholest-7-en-3[beta]-ol (lathosterol), lanosterol,
[beta]-sitoserol, ergosterol, cholest-4,6-dien-3-one, coprostan3-ol,
25-hydroxycholesterol, cholesterol-5[alpha],6[alpha]-epoxide,
4-cholesten-3-one, 5-cholesten-3-one, and dihydrocholesterol. In the GC
IDMS method none of the steroids studied interfered, as was expected
from earlier studies [12]. In the LC IDMS only lathosterol with a
retention time of 5.49 min and a molecular mass of 386.7 could interfere
with the determination of the unlabeled cholesterol, the actual signal
of lathosterol at m/z = 386 being 30.1% of that of an equal amount of
cholesterol. Coprostan-3-ol with a retention time of 5.28 min and a
molecular mass of 388.7 could interfere with the determination of the
labeled cholesterol, the signal of coprostan-3-ol at m/z = 386 being
1.9% and at m/z = 389 8.9% compared with that of an equal amount of
cholesterol. All other steroids tested could not interfere, because they
eluted well separated from the cholesterol in HPLC.

MEMORY EFFECTS

We tested the analytical systems used for LC IDMS and GC IDMS for
the existence of memory effects. If an unexpected memory effect were
present, then the measured isotope ratio of a sample would be influenced
by the history of samples measured previously. So we routinely measured
on both systems sequences of five determinations of a sample of
unlabeled cholesterol, followed by five determinations of the
isotope-labeled cholesterol and repeated this all five times. We never
observed a drift in the isotope ratios for the unlabeled or the labeled
cholesterol with both IDMS methods. If a memory effect were present,
then it should be detectable at least in this situation, measuring a
sequence of samples with the most extreme isotope ratios possible.

LINEARITY OF THE LC IDMS METHOD

We tested the linearity of the relationship between the mass ratios
(c/[c.sup.*]) of unlabeled and labeled cholesterol and the isotope
ratio, calculated from the area [A.sub.386] under the peak obtained in
the SIM chromatogram at m/z = 386 and from the area [A.sub.389] under
the peak obtained in the SIM chromatogram at m/z = 389. The isotope
ratio was corrected for the isotope ratio [f.sub.1] = 0.00646 of the
pure labeled cholesterol and the isotope ratio [f.sub.2] = 0.00242 of
the pure unlabeled cholesterol, both determined in the previous section,
by the formula

The linearity was tested in the range of mass ratios (c/[c.sub.*])
of unlabeled and labeled cholesterol between 0.25 and 2.0. The data,
shown in Fig. 2, demonstrate that linearity is given for isotope ratios
between 0.25 and 2.0. The relationship is Y = (-0.0005 [+ or -] 0.0116)
+ (1.1404 [+ or -] 0.0094) x (c/[c.sup.*]) with a correlation
coefficient of r2 = 0.9998, the intercept of the regression line is not
significantly deviating from the origin, and there was no intrinsic
nonlinearity observable in the range of mass ratios tested.

STANDARD STABILITY

The stability of the mass ratio between days was calculated from
the measured, corrected isotope ratios Y found for the lower and for the
higher standard. The statistic was done for the first measurement of the
standards on each day. For the lower standard we found for 20 days a
mean of 0.7514 and a SD of 0.0021 (CV = 0.28%); for the higher standard
we found under the same conditions a mean of 1.2529 and a SD of 0.0033
(CV = 0.26%).

[FIGURE 2 OMITTED]

STANDARD CONSISTENCY

For the assessment of the standard consistency we used every day
two sets of two standards, one set for the calibration of the method and
an independent second set with the same isotope ratios for measuring
standard recovery. The second set of standards was treated like unknown
samples, and the results were calculated as for unknown samples. The
data were evaluated as described elsewhere [5]. In the LC IDMS we never
found differences between the calculated mass ratio (c/[c.sup.*]) of
unlabeled and labeled cholesterol and the weighed-in mass ratio
(c/[c.sup.*]) of unlabeled and labeled cholesterol >0.20%.

SAMPLE PREPARATION

The hydrolysis procedure used in this work is similar to that
described elsewhere [6,10]. The main difference is the much larger
excess of hydrolyzing reagent used here. So we only checked whether the
simultaneous doubling of the hydrolyzing reagent and the hydrolysis time
had an impact on the results obtained. Each sample was hydrolyzed in a
separate experiment with a doubled amount of hydrolyzing reagent and was
incubated for 6 instead of 3 h. In no case could a difference be
observed between the results obtained for both hydrolysis procedures.

We also checked for the possible influence of the sample
preparation method on the standards. We performed experiments in which
we prepared standards like unknown samples. No differences could be
observed between the measured isotope ratios of the standards used for
calibration and prepared normally and the standards prepared like serum
samples or control materials.

PRECISION AND ACCURACY OF THE LC IDMS METHOD

Table 1 shows the day-to-day precision for five different
serum-based control materials, obtained from 10 independent series on
separate days. For the three control materials obtained from NIST, the
CVs are <1.0%. For the other control materials the imprecision is
slightly >1.0%. This could be caused by the vial-to-vial
variabilities of the control material rather than by LC IDMS method
itself. The control materials obtained from NIST were also used as
controls for the GC IDMS method. In that case CVs <1.0% were obtained
also.

The accuracy of the LC IDMS method for the determination of total
cholesterol in serum was checked with the five different serum-based
control materials, for which the target values were based on Definitive
Methods in the case of the NIST materials and on GC IDMS Reference
Method-based target values supplied by the manufacturer. For all tested
control materials the bias (from 10 independent series on separate days)
was <1.0% and in all cases lower than the CV. In the GC IDMS method,
the bias of the three control materials SRM 909, SRM B1, and SRM B2 was
<1.0% as well.

For the method comparison 28 pooled human sera were used and
measured independently with the LC IDMS method presented and GC IDMS as
a Reference Method. Fig. 3 shows the CVs, obtained for each serum pool
from five determinations with both methods. The mean imprecision was
0.66% (range 0.26-1.21%) for the GC IDMS method and 0.72% (range
0.31-1.17%) for the LC IDMS method. The mean results for the 28 pooled
human sera, obtained by our LC IDMS method, were compared with the
chosen GC IDMS Reference Method. Fig. 4a shows the correlation of the
results including the graph of the following linear relationship
obtained by the method of Passing and Bablok [13]: [C.sub.Lc IDMs] =
0.993 X [C.sub.GC IDMs] - 0.15 mg/L; 95% confidence interval for the
slope: 0.978-1.008; 95% confidence interval for the intercept: -25.8
mg/L to +25.9 mg/L; correlation coefficient r: 1.000; standard error of
the estimates [S.sub.y|x]: 1.375.

[FIGURE 3 OMITTED]

In Fig. 4b the relative deviations of the results obtained with the
LC IDMS method from those obtained with the GC IDMS Reference Method
[11] are presented graphically. For 32% of the samples the relative
deviation between the LC IDMS and the GC IDMS method was <0.5%; for
93% of the samples the relative deviation did not exceed 2.0%.

Discussion

The aim of this study was the evaluation of an isotope dilution
method for the determination of total cholesterol in serum by
HPLC-particle beam-El-mass spectrometry as an analytical method. This
type of coupling liquid chromatography to mass spectrometry has not been
used for the development of isotope dilution methods before. HPLC
separations of cholesterol have been described before [10], so the only
problem was the compatibility of the eluent system with a particle-beam
interface used for coupling the liquid chromatograph with the mass
spectrometer [14]. After optimization of the pneumatic and thermal
variables of the particle-beam interface, the El ionization was
sensitive enough for the detection of the underivatized cholesterol. As
has been already expected from the results presented [10], the peak of
the cholesterol in SIM chromatograms obtained by the LC IDMS is much
broader compared with that obtained by GC IDMS. On the other hand, in
both methods neither the peak shape nor the baseline noise deteriorated
the quality of the quantification process. A great disadvantage of the
LC IDMS is that much more analyte has to be introduced in the
instrument. This is most likely because of the particle-beam interface
and will limit the range of applicability of the LC IDMS approach
presented in this work. The study of possible interference of exogenous
or endogenous steroids showed that the cholesterol precursor lathosterol
is the most interfering steroid, but its concentration in serum is
<0.3% of the concentration of the cholesterol itself [15]. Because
the relative intensity of lathosterol at m/z = 386 is only 30% of that
obtained for an equal amount of cholesterol, this interference is not of
practical significance. Coprostanol had even a lower relative intensity
compared with cholesterol and, like lathosterol, has not been reported
to be present in concentrations in serum that would alter the results
obtained by the CDC Reference Method for cholesterol [16].

[FIGURE 4 OMITTED]

As has been stated X10], the use of an isotope-labeled internal
standard differing only by 2 amu from the unlabeled cholesterol leads to
a nonlinearity between the corrected peak intensity ratio Y and
(c/[c.sup.*]). The use of an internal standard differing by 3 amu from
the unlabeled compound prevents this effect nearly completely.

We chose two standards and bracketing as the calibration method, as
described previously [6]. The use of two standards proved to be
sufficient, the standards were consistent within-run, and the isotope
ratios obtained for the standards were stable for at least 1 month with
a CV <0.3% in the measured isotope ratios. The sample preparation
technique did not influence the measurement of isotope ratios of
standards prepared like serum samples. As stated earlier [6], even
shorter periods of time for hydrolysis and lower concentrations of the
hydrolyzing reagent lead to a total hydrolysis of cholesterol esters. We
did not observe any differences between aliquots hydrolyzed under
different conditions. The error introduced by the uncertainty of the
used standard materials as well as the error introduced by the weighing
procedure are in the order of magnitude of 0.1-0.2%. Overall the
imprecision of the LC IDMS method was [less than or equal to] 1.2% for
all control materials studied, <1% for NIST materials. For all
control materials the bias was lower than the CV of the results.
Furthermore in the case of the NIST control sera with certified
uncertainty of the target value, the bias found was lower than this
uncertainty, which represents the highest bias allowed (Table 1).

The new method was tested against an GC IDMS Reference Method for
the determination of total cholesterol in serum. For 28 pooled human
sera we found a very good correlation between the results obtained with
the new method and the Reference Method. For 93% of the samples the
difference of the results for both analytical methods did not exceed
2.0%. Neither the imprecision nor the bias between the methods depended
on the concentration of the pooled serum samples used. Even for the
highest concentration sample (4351 mg/L) measured by LC IDMS, the bias
was 2.04% and was therefore not excluded from the statistical analysis;
the relevance of a precise measurement in this range of concentrations
is nevertheless limited. The between-run imprecisions for these serum
pools did not exceed those obtained for the control materials.

In this study a new analytical technique was used for the
development of an isotope dilution method. The liquid chromatographic
separation method is applicable to a wide variety of dissolved organic
molecules [14], and no derivatization procedure is required previous to
analysis, making the sample preparation more simple than in the GC-MS
method. The comparison of accuracy and precision of the new LC IDMS
method with an GC IDMS method chosen as reference did not show any
substantial advantage of the GC IDMS method. The method presented in
this study may be considered as a prototype of isotope dilution methods
applicable to analytes for which the sample preparation required for gas
chromatography is complicated or a volatile derivative does not exist.

References

[1.] Boutwell JH, ed. Proceedings of a conference on national
understanding for the development of reference materials and methods in
clinical chemistry. Washington DC: AACC, 1978.

[3.] NRSCC1-T. Tentative guidelines for the development of
Definitive Methods in clinical chemistry for the National Reference
System in Clinical Chemistry. Villanova, PA: National Committee for
Clinical Laboratory Standards, 1982.